Cluster analysis of insulin action in adipocytes reveals a key role for Akt at the plasma membrane.

Diabetes and Obesity Research Program, The Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia.

Abstract

The phosphatidylinositol 3-kinase/Akt pathway regulates many biological processes, including insulin-regulated GLUT4 insertion into the plasma membrane. However, Akt operates well below its capacity to facilitate maximal GLUT4 translocation. Thus, reconciling modest changes in Akt expression or activity as a cause of metabolic dysfunction is complex. To resolve this, we examined insulin regulation of components within the signaling cascade in a quantitative kinetic and dose-response study combined with hierarchical cluster analysis. This revealed a strong relationship between phosphorylation of Akt substrates and GLUT4 translocation but not whole cell Akt phosphorylation. In contrast, Akt activity at the plasma membrane strongly correlated with GLUT4 translocation and Akt substrate phosphorylation. Additionally, two of the phosphorylated sites in the Akt substrate AS160 clustered separately, with Thr(P)-642 grouped with other Akt substrates. Further experiments suggested that atypical protein kinase Czeta phosphorylates AS160 at Ser-588 and that these two sites are mutually exclusive. These data indicate the utility of hierarchical cluster analysis for identifying functionally related biological nodes and highlight the importance of subcellular partitioning of key signaling components for biological specificity.

Lack of correspondence between Akt phosphorylation and GLUT4 translocation. Insulin dose-response of Ser(P)-473 Akt from the TCL and HA-GLUT4 translocation to the PM in 3T3-L1 adipocytes are shown. HA-GLUT4-expressing 3T3-L1 adipocytes were serum-starved for 2 h and stimulated with indicated dose of insulin for 20 min. Total cell lysates were immunoblotted with Ser(P)-473 Akt antibody. The amount of HA-GLUT4 at the plasma membrane was determined by anti-hemagglutinin fluorescence immunolabeling of nonpermeabilized cells and expressed as percentage of total cellular HA-GLUT4, determined by labeling of permeabilized cells. This was then normalized, such that 0 represents the basal level, and 1 represents the maximal response. (n = 3).

Dose response and time course of insulin and PDGF signaling in 3T3-L1 adipocytes.A, dose response of insulin signaling. 3T3-L1 adipocytes were serum-starved for 2 h and treated with different doses of insulin for 20 min. Total cell lysates were immunoblotted with antibodies raised against a panel of insulin-signaling components. Immunoblots are from a representative experiment (n = 7–9). B, signaling components were assigned into two different clusters based on their dose-response profiles. The x axis is log10 of the insulin dose, and the y axis is percentage of maximum response normalized to 100 nm insulin. Results are displayed as means ± S.D. (n = 7–9). C, time course of insulin and PDGF signaling. 3T3-L1 adipocytes or human PDGF receptor expressing 3T3-L1 adipocytes were serum-starved for 2 h and treated with either 100 nm insulin or 20 ng/ml PDGF for indicated periods of time. Total cell lysates were immunoblotted with antibodies raised against a panel of insulin-signaling components. Immunoblots are from a representative experiment (Insulin time course n = 3; PDGF time course n = 2). D, time course of various insulin-signaling components expressed as a maximum response of 30 min insulin stimulation. Results are displayed as means ± S.D.

Cluster analysis of dose-response and/or kinetics of insulin- and PDGF-stimulated Akt signaling.A, clustering of insulin response over dose variables. The two major clusters are shown as follows: Akt substrates and HA-GLUT4 at the PM (black) and other signaling components (gray). B, clustering of insulin response over dose and temporal variables. The two major clusters are shown as follows: Akt substrates and HA-GLUT4 at the PM (black) and other signaling components (gray). Note these are identical in grouping to that of A. C, clustering of insulin response over temporal variables. Two major groups of phosphorylated signaling components were found, shown in light gray and dark gray, along with two outliers. The two groups from the dose variable clustering in A and B are shown in black and gray on the component labels. D, clustering of PDGF response over temporal variables. As in C, two major groups of phosphorylated signaling components were found, shown in light gray and dark gray, along with two outliers. The only difference in grouping to C is that of Thr(P)-1462 TSC2. Again, the two groups from the dose variable clustering in A and B are shown in black and gray on the component labels.

Akt at the plasma membrane correlates with GLUT4 trafficking.A, insulin dose response of Ser(P)-473 Akt at the cell surface. 3T3-L1 adipocytes were serum-starved for 2 h and treated with different doses of insulin for 20 min. Cells were fixed and immunolabeled with Ser(P)-473 Akt followed by Cy2-conjugated secondary antibody. Confocal slice images were taken at the base of the cell. Images are from a representative experiment (n = 3; 30–50 images per condition in each experiment). Scale bar, 40 μm.B, insulin dose-response of Ser(P)-473 Akt at the cell surface. 3T3-L1 adipocytes were serum-starved for 2 h and treated with different doses of insulin for 20 min. Cells were fixed and immunolabeled with Ser(P)-473 Akt followed by Alexa488-conjugated secondary antibody. Cells were then imaged by TIRFM. Representative images are shown (n = 2; 30–50 images per condition in each experiment). Scale bar, 10 μm.C, 3T3-L1 adipocytes were serum-starved for 2 h and treated with either 0.1% DMSO or 10 μm Akti-1/2 for 15 min before stimulation with 10 nm insulin for 20 min. Cells were fixed, and active Akt was immunolabeled with anti-Ser(P)-473 Akt antibody in combination with Cy2-conjugated secondary antibody in permeabilized cells. Images were taken as in A. Images are from a representative experiment (n = 3; 30–50 images per condition in each experiment). Scale bar, 40 μm.D, quantification of C. The amount of Ser(P)-473 Akt at cell surface was determined using the region detector program. Results are displayed as means ± S.D. *, p < 0.05, insulin-treated versus insulin and Akti-1/2 treatment, Student's t test (n = 3). Inset is the total cell lysates of 3T3-L1 adipocytes immunoblotted with antibodies against Ser(P)-473 Akt and total Akt. E, insulin dose response of Ser(P)-473 Akt from the TCL, IF-PM, and TIRFM-PM expressed as a maximum response of 100 nm insulin stimulation. Results are displayed as means ± S.D. F, clustering of insulin response over dose variables for signaling components from the TCL (Thr(P)-308 Akt, Ser(P)-473 Akt, Ser-588 AS160, and Thr-642 AS160), Ser(P)-473 (IF-PM), Ser(P)-473 (TIRFM-PM), and HA-GLUT4 (PM). Note that although Ser(P)-588 AS160 is clustered with the TCL Akt components, Thr(P)-642 AS160 clusters with both the Akt components at the PM as well as HA-GLUT4 at the PM.

Differences in phosphorylation in Ser-588 and Thr-642 on AS160.A, 3T3-L1 adipocytes were serum-starved for 2 h and treated with 100 nm insulin for 20 min. Cell lysates were prepared, and Thr-642-phosphorylated AS160 was immunoprecipitated (IP) with specific Thr(P)-642 AS160 rabbit antibody. A nonimmunized rabbit IgG (CT) was used as control for immunoprecipitation using insulin-stimulated lysates. The immune complexes (IP) and the flow-through (FT) were then immunoblotted with Ser(P)-588 AS160, Thr(P)-642 AS160, and total AS160 antibodies. TCL were also immunoblotted for Ser(P)-588 AS160, Thr(P)-642 AS160, total AS160, Ser(P)-473 Akt, and total Akt. B, HEK 293 cells were transfected with either V5 PKCζ A119E (dominant active) or V5 PKCζ KW (dominant negative), in combination with different mutants of FLAG-tagged AS160 (S318A, S341A, S588A, T642A, 4P, or wild type (wt)) or FLAG vector control. Cell lysates were prepared, and FLAG AS160 wild type or mutants or FLAG control were immunoprecipitated with FLAG antibody. The immune complexes were then immunoblotted with FLAG, V5, and phospho-Ser PKC substrate antibodies. WB, Western blot. C, quantification of B. The amount of phosphorylation was normalized against immunoprecipitated FLAG AS160. Results are displayed as means ± S.D. (n = 2). WT, wild type.

Phospho-AS160 is concentrated in a detergent-insoluble fraction.A, 3T3-L1 adipocytes were serum-starved for 2 h and treated with 100 nm insulin for the indicated period of times. Cells were then fractionated into Triton-soluble fraction (Txsol) or Triton-insoluble fraction (Txinsol) fractions. TCL and the fractions were then subjected to Western blot analysis with Ser(P)-473 Akt, total Akt, Ser(P)-588 AS160, Thr(P)-642 AS160, total AS160, Ser(P)-9 GSK3β, total GSK3β, caveolin, Crk, and 14-3-3β antibodies. B, quantification of A. Specific activity of Ser(P)-473 Akt, Ser(P)-588 AS160, Thr-642 AS160, and Ser(P)-9 GSK3β from various fractions normalized against insulin stimulation (30 min) of the TCL. Results are displayed as means ± S.D. (n = 2).